Draper Michael P, August Paul R, Connolly Timothy, Packard Brian, Call Katherine M
Paratek Pharmaceuticals, 75 Kneeland Street, Boston, Massachusetts 02111, USA.
Genomics. 2002 Apr;79(4):603-7. doi: 10.1006/geno.2002.6738.
The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries.
生成和获取全长（FL）cDNA的能力对于基因组学领域至关重要。传统cDNA文库中的大多数cDNA缺乏起始的5' ATG，这使得难以获得FL克隆。我们在此报告一种改进的制备富含FL cDNA文库的方案。我们证明，如果在cDNA合成中使用高质量RNA，则可以为长度超过7 kb的转录本生成高质量的FL cDNA。此外，我们证明了大小分级分离作为一种手段的实用性，可用于产生含有高比例起始5' ATG且插入片段大小大于4 kb的克隆的文库。该方法简单、成本效益高，并且可以在大多数具备分子生物学操作能力的实验室中进行。最后，用于分析cDNA和文库的新方法应该对其他致力于创建高质量cDNA文库的人有用。
